Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells
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Systemic transfer.pdf
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Author(s)
Stanton, Brynne C.
Siciliano, Velia
Wroblewska, Liliana
Clancy, Kevin
Trefzer, Axel C.
Chesnut, Jonathan D.
Weiss, Ron
Voigt, Christopher A.
Ghodasara, Amar Navin
Date Issued
October 2014
Journal
ACS Synthetic Biology
Publisher
American Chemical Society (ACS)
Citation
Stanton, Brynne C., Velia Siciliano, Amar Ghodasara, Liliana Wroblewska, Kevin Clancy, Axel C. Trefzer, Jonathan D. Chesnut, Ron Weiss, and Christopher A. Voigt. “Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells.” ACS Synthetic Biology 3, no. 12 (December 19, 2014): 880–891. © 2014 American Chemical Society
Version
Final published version
Abstract
Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10[superscript 5] members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
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Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
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DOI of Published Version
https://doi.org/10.1021/sb5002856
